1. Field of the Invention
This invention relates to specific binding fluoroassay assay techniques. More particularly, it relates to homogeneous specific binding fluorassay techniques which employ water-soluble rare earth metal chelate fluorophores together with fluorescence measurements which eliminate or reject interferring background fluorescence and permit direct and highly sensitive determination of analytes in aqueous media.
Specific binding assay techniques are useful for determining various substances of diagnostic, medical, environmental and industrial importance which appear in liquid media at very low concentrations. Specific binding assays are based on a specific interaction between the analyte under determination and a binding partner therefor. Where one of the analyte and its binding partner is an antibody and the other is a corresponding hapten or antigen, the assay is known as an immunoassay.
In such assays, it is necessary to have a tag or label present which can give a measurable indication of the extent to which the specific binding reaction takes place. For example, fluorescent labels can be present on the binding partner and the incorporation of this fluorescent label into the specifically bound pair can be used to detect formation of the pair. Alternatively, the specific binding reaction can give rise to a change in the fluorescence properties of the fluorophore. As outlined by I. Wieder in U.S. Pat. No. 4,058,732, the detection of this fluorescence can be made more sensitive by employing time-gated detection to reject short-lived background fluorescence and more accurately detect long-lived fluorescence such as emitted by rare earth metal chelates. These background rejection techniques have been employed with advantage heretofore in a variety of heterogeneous analysis methods. A heterogeneous assay is one in which the fluorescently labeled pair is physically separated from unbound fluorophores.
The present invention provides techniques in which the background rejection methods are applied to homogeneous assays. A homogeneous assay is one in which the fluorescently labeled pairs are not separated from the unbound fluorophores but the bound fluorophores and unbound fluorophores are distinguishable from one another. See J. BIOL. CHEM., 251, 4172-8 (1976).
2. Description of the Prior Art
U.S. Pat. No. 4,058,732, issued on Nov. 15, 1977 to Irwin Wieder describes the use of fluorescent background rejection in assay techniques.
U.S. Pat. No. 4,341,957, issued to Irwin Wieder describes antibodies coupled to fluorescent rare earth chelates.
U.S. Pat. No. 4,352,751 issued on Oct. 5, 1982 to Wieder and Wollenberg discloses a family of rare earth metal chelates and their use in fluorescent background rejection techniques. Similarly, European Patent Application 68875 A2 filed on Jun. 28, 1982 of Eastman Kodak Co. discloses fluorescent rare earth chelate labels in fluoroassay techniques.
European Patent Application 64484 A2 of Wallac Oy discloses a heterogeneous assay method wherein a lanthanide chelate is coupled to a specific binding substance, reacted in a binding reaction and isolated from the binding reaction reaction mixture by physical separation. The lanthanide ion label is then released from the chelate by an acid treatment and measured using the Wieder fluorescence background rejection technique. Other references of interest include articles appearing at CLIN. CHEM., Winston Salem, N.C., 29(1) 65-8 (1983) and PROC. NATL. MEET. BIOPHYS. MED. ENG. FINL., 4, 199-202 (1982) and U.S. Pat. No. 4,374,120 all from the Wallac Oy group: and Kodak's U.S. Pat. No. 4,283,382, issued Aug. 11, 1981.
A recent item of interest is European Patent Application 103558 published Mar. 21, 1984 in which the Wallac Oy group proposed the use of fluorescence background rejection in certain homogeneous assay settings. However, the method shown in that patent application utilizes reagents that are normally insoluble and weakly fluorescent in water and employs complicated micelle formation techniques to enable the use of these reagents with aqueous test media. This inability to operate directly in aqueous media is a serious failing since virtually every important specific binding reaction is identified primarily in aqueous systems.
Other art to be noted, but probably less in point, includes the article "A Review of Fluoroimmunoassay and Immunofluorometric Assay", D. S. Smith et al, ANN. CLIN. BIOCHEM., 18, (1981) 253-274 which provides a summary of the heterogeneous and homogeneous fluoroassay techniques proposed heretofore; U.S. Pat. Nos. 3,998,943; 4,020,151; 3,939,350; 4,220,450 and 3,901,654 which show analytical assay techniques.
There are two general types of homogeneous fluoroassays. In the more common of these, the bound fluorophores have a decreased or "quenched" fluorescence as compared to the unbound fluorophores so that the extent of binding can be detected by a decrease in fluorescence. A variety of quenching assays have been proposed. (See, for example, Shaw, et al, J. CLIN. PATHOL. 1977, 30, 526-31; Broughton, et al, CLIN. CHEM. 1978, 24, 1033; Shaw, et al, CLIN. CHEM. 1979, 25, 322-5; Dandliker, et al, IMMUNOCHEMISTRY 1970, 7, 799-828; Nargessi, et al, J. IMMUNOL. METH. 1979, 26, 307-313; Lopatin, et al, BIOCHEMISTRY 1971, 10, 208-13; Portmann, et al, IMMUNOCHEMISTRY 1975, 12, 461-6; Levison, et al, BIOCHEMISTRY 1970, 9, 322-31; Voss, et al, IMMUNOCHEMISTRY 1976, 13, 447-53; Nargessi, et al, CLIN CHEM ACTA 1978, 89, 461-7; Zuk, et al, CLIN CHEM 1979, 25, 1554-60 and Brinkley, et al, CLIN CHEM 1979, 25, 1077.) Similarly, SYVA corporation has proposed a fluorescein-rhodamine homogeneous assay system in which the fluorescein is affixed to the antigen and the rhodamine is affixed to the antibody. When these two groups are brought together, quenching results.
In the other type of homogeneous fluoroassay, the bound fluorophore has an increased or "enhanced" fluorescence as compared to the unbound fluorophore. See D. S. Smith, FEBS LETT., 77, 25-7, (1977) in which a fluorescein-labeled T.sub.4 was used to accomplish an enhancement.
The foregoing techniques have been based in general upon the use of rhodamines and fluorescein, for the most part, and have not been based upon materials such as the rare earth chelates which are particularly advantageous in the setting of fluorescence background rejection. Notwithstanding the recognized advantages of homogeneous assays and of fluorescence background rejection as analysis techniques there has not yet been provided the methodology to merge these two technical advances in a general practical way so as to effectively analyze the wide range of extremely dilute aqueous solutions which the field provides. More particularly, there has been no effective application of these techniques to provide an enhancement fluorescence assay. This failure is especially important because a main field of interest involves samples which have concentrations of analyte less than 10.sup.-8 molar, especially in the 10.sup.-8 to 10.sup.-14 molar range and below, which is pertinent to determining natural products, hormones, serum proteins, and the like. It is the primary object of this invention to provide practical homogeneous assays for analytes in dilute aqueous media using fluorescence background rejection as the detection event.